- Email: [email protected]
Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters
Vaccine 23 (2005) 2862–2868
Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters夽 Giuliana Magria , Mario Clericia,∗ , Paola Dall’Arab , Mara Biasina , Maria Caramellic , Cristina Casalonec , Maria Laura Gianninob , Renato Longhid , Luca Piacentinia , Silvia Della Bellae , Paola Gazzuolac , Piera Anna Martinob , Silvia Della Bellaa , Claudia Pollerab , Maria Puricellib , Francesco Servidab , Ines Crescioc , Adriano Boassoa , Wilma Pontib , Giorgio Polib a
Laboratory of Immunology, DSP LITA Vialba, Universita’ degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy b Dipartimento di Patologia Animale, Igiene e Sanit` a Pubblica Veterinaria, Sezione di Microbiologia e Immunologia, Universit`a degli Studi di Milano, Centro di Eccellenza sulle Malattie Neurodegenerative, Milan, Italy c Centro per lo Studio e le Ricerche sulle Encefalopatie Animali e Neuropatologie Comparate, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D’Aosta, Torino, Italy d Istituto di Biocatalisi e Riconoscimento Molecolare, C.N.R., Milano, Italy e The Immunology Department of Biomedical Sciences and Technologies, University of Milan, Milan, Italy Received 10 May 2004; received in revised form 12 October 2004; accepted 25 November 2004 Available online 31 December 2004
Abstract Effective therapy for prion diseases is currently unavailable. Recently, vaccination was shown to be effective in mouse models of a particular neurodegenerative conditions: Alzheimer’s disease (AD). Here, we report that vaccination with synthetic oligopeptides homologous to the hamster (Mesocricetus auratus) prion protein augments survival time in animals infected intraperitoneally with 263K scrapie agent. For each hamster included in the study, prion-specific serum antibodies as well as deposition of pathological prion protein (PrPres ), glial fibrillary acidic protein (GFAP), and mRNA expression for cytokines (TNF␣, IL-1, IL-10) in brain tissues were evaluated. In immunized animals, increased survival after challenge was associated with a reduction of cerebral lesion, PrP deposition and GFAP expression; in these animals, anti-prion protein peptide antibody levels were increased, and the expression of pro-inflammatory cytokines (TNF␣ and IL-1) was reduced. Vaccination could be an effective therapeutic approach to postpone disease onset. © 2004 Elsevier Ltd. All rights reserved. Keywords: Prion; Vaccination; Inflammation
1. Introduction Prion diseases, or transmissible spongiform encephalopathies (TSE), are rare, progressive, fatal neurode夽 Supported by grants from ISS: “Programma Nazionale di Ricerca sull’
AIDS” and “Progetti strategici: fattori genetici, patogenetici e biochimici responsabili della sensibilit`a/resistenza alle EST”, by Centro di Eccellenza CISI, and by the Japan Health Science Foundation. ∗ Corresponding author. Tel.: +39 02 5031 9679; fax: +39 02 5031 9677. E-mail address: [email protected] (M. Clerici). 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.11.067
generative conditions that include Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats [1,2]. These diseases are characterized by spongiform degeneration of the brain, accompanied by appearance of activated microglia and astrocytes as shown by the altered expression of the glial fibrillary acidic protein (GFAP), a marker of astrocytosis [3–5]. The hallmark of TSE diseases is the conversion of normal cellular prion protein (PrPc ) into an infectious disease-associated protease-resistant isoform (PrPres ) that accumulates primarily in the brain
G. Magri et al. / Vaccine 23 (2005) 2862–2868
and in the lymphoreticular system [6]. PrP deposits are also associated with a chronic inflammatory response mainly based on cytokine production (TNF-␣, IL-1, IL-6) [7–10]. Prion diseases share important clinical, neuropathological, and cell biological features with another, more common degenerative cerebral disease. Both AD and prion diseases are characterized by a cascade of events that terminate with the extracellular deposition of amyloid- (A) or PrPres fibrillar peptides, respectively [11–14]. Currently, none of the prion diseases has an effective treatment [15]. In spite of this, recent evidences suggest that immunotherapeutical intervention could be worth pursuing [16]. It has recently been shown that immunization of transgenic mice with AD-related neuropathology using fibrillar A1–42 as an antigen, reduces or prevents cerebral A amyloid deposits [17,18]. An antibody-mediated response is likely to be critical for a therapeutic response, since analog results have been obtained with passive immunization [19]. Similar approaches for prion diseases have recently shown that: (1) anti-PrP monoclonal antibodies or fragment antigen binding (Fab) with little or no affinity for PrPres can inhibit PrPres formation in a dose-dependent manner [20,21]; (2) coexpression of anti-PrPc antibodies (6H4) and PrPc in transgenic mice (Prnp+/− -6H4) does not induce an obvious autoimmune disease and confers protection from scrapie [22]; and (3) immunization of wild-type mice with prion protein peptides induces anti-PrP antibodies and reduces PrPres formation in scrapie-infected neuroblastomas transplanted into immunized mice [23]. We investigated whether active immunization with synthetic peptides of prion protein could influence the progression of prion disease in a hamster model of scrapie.
2. Material and methods 2.1. Conjugate preparation, vaccination and infection protocol Thirty Female Golden Syrian hamsters, 3–4 weeks old (Charles River, Como, Italy) were used according to the EU International Guidelines for animal experimentation. The animals were randomly divided into three groups (six animals/group). A negative control group of animals (NC) treated with adjuvant alone and a positive control group (PC) (infected but non-immunized animals) were also included in the study. Three peptides (peptide A: p105–128: cPKTNMKHMAGAAAAGAVVGGLGGY; peptide B: p119–146: cGAVVGGLGGYMLGSAMSRPMMHFGNDWE; peptide C: p142– 179: GNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDC) homologous to the hamster prion protein and resembling different fragments (30–32 amino acids) of the molecule were used. The choice of these three peptides was made because they cover the N-terminal (A peptide)
2863
and the central (B and C peptides) sequence of hamster PrP. In particular, B and C peptides contains the protein domain possibly involved in PrP conversion process. Peptides were synthesized by the solid phase Fmoc method [24] using an Applied Biosystem model 433A Peptide Synthesizer (Foster City, CA, USA). To the peptides not incorporating thiolfunction, cysteine was added at the amino termini or at the carboxy termini of the peptide to enable specific conjugation to the carrier protein. After peptide assembly, peptides were cleaved from the resin by treatment with a mixture of 80% trifluoroacetic acid, 5% water, 5% phenol, 5% thioanisole, 2.5% ethandithiol and 2.5% triisopropylsilane (reagent K) [25] for 3 h at room temperature, with concomitant side chain deprotection. Peptides were lyophilized and than analyzed and purified to apparent homogeneity by reversed phase high performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the peptides was performed on a Voyager-RP Biospectrometry Workstation (PE Biosystem Inc.). Observed experimental values for peptide masses were in agreement with theoretical calculated values. Peptides were specifically conjugated to freshly prepared maleimido-activated Mariculture Keyhole Limpet Hemocyanin (mcKLH), using sulfosuccimnimidyl 4(N-maleimidomethyl)cyclohexane-1-carboxylate (SulfoSMCC), both from Pierce, according with the procedure described by Liu et al. [26], diluted in pyrogen free saline solution mixed with an equal volume of complete (first vaccination) or incomplete (booster vaccinations) Freund’s adjuvant immediately before administration. Animals were inoculated distributing 0.5 ml of the suspension containing a total amount of 10 g of different peptides both by intramuscular route into right and left popliteus and by subcutaneous and intradermic route in the right and left dorsal region. Animals were immunized with three injections at 10 days interval before infection (T0, T11, T20), challenged by intraperitoneal route with 105 ID50 of 263K hamster adapted scrapie strain (T25) and then immunized with other five injections at 20 days interval post-infection (T28, T62, T81, T103, T126). 2.2. Antibody response ELISA 96-well microplates (Maxisorp, Nunc, Roskilde, Denmark) were coated for 18 h at 4 ◦ C with 100 l/well of free peptides, diluted in carbonate–bicarbonate buffer at the concentration of 250 ng/ml. After four washings with PBS-Tween 0.05% (PBS-T), 100 l/well of 1:100 pre-diluted serum samples were added and the plates incubated for 1 h at 37 ◦ C. After four PBS-T washings, 100 l/well of 1:500 prediluted horseradish peroxidase-conjugated anti-hamster IgG (ICN Biomedicals, Cappel, Irvine, CA, USA) were added and the plates incubated for 1 h at 37 ◦ C. After four more
2864
G. Magri et al. / Vaccine 23 (2005) 2862–2868
PBS-T washings, 100 l of ABTS substrate was added to each well. The reaction was stopped by the addition of 50 l of 1% SDS/well and the plates were read at 405 nm using the Labsystem Multiskan EX (Helsinki, Finland). 2.3. Histological examination The whole brain was collected. Each specimen was cut in half longitudinally along the midline; one half was fixed in 10% formol saline and decontaminated with 96% formic acid for 1 h. The unfixed portion was frozen at −80 ◦ C. Four transverse sections were obtained from each specimen, paraffin embedded, cut in serial sections (5–6 m), and stained with haematoxylin and eosin by standard procedures. Ten areas of the grey matter were examined in four neuroanatomical locations (cerebral cortex septal nuclei of telencephalon; cerebral cortex, hypothalamus, thalamus, hippocampus of diencephalon; tectum of midbrain and substantia nigra of mesencephalon; cerebellar cortex of the folia and medulla) [27]. The lesion profile method was applied to each area, by two independent observers, giving a score from 0 to 4 depending on the density of vacuolation and spongiosis. 2.4. Immunohistochemical analysis: PrPres and GFAP staining Five-micrometer thick tissue sections were de-waxed and re-hydrated by routine methods. For PrPres , the sections were immersed in 98% formic acid for 20 min, washed in distilled water, and autoclaved for 20 min at 121 ◦ C in distilled water. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide for 20 min at room temperature. For gfap endogenous peroxidase activity was blocked in 3% hydrogen peroxide for 20 min at room temperature and subjected to an antigen retrieval procedure with two 5 min 850 W microwave oven passages in 0.01 M (pH 6) citrate buffer. To remove non-specific tissue antigens, the sections were incubated with 5% normal goat serum for 20 min at room temperature. Incubation with the primary antibody 1/IST [28] raised in rabbit against 263K Scrapie strain, diluted 1:250, and anti-gfap policlonal antibody (Dako, Carpinteria, CA) diluted 1:1000 was performed overnight at 4 ◦ C. The rest of the immunohistochemical procedure was carried out by a commercial immunoperoxidase technique (Vectastain ABC kit, Vector Burlingame, CA), using 3,3 diaminobenzidine (Dako, Carpinteria, CA) as chromogen and the sections were counterstained with Meyer’s haematoxylin. For PrPres , 13 areas in four neuroanatomical location were examined (cerebral cortex, sub-ependymal area, submeningeal area and septal nuclei of telencephalon; cerebral cortex, hypothalamus, thalamus, hippocampus of diencephalon; tectum of midbrain, sub-ependymal area, and substantia nigra of mesencephalon; cerebellar cortex of the folia and medulla); for GFAP, seven sites in four neuroanatomical
area were examined (cerebral cortex of telencephalon; cerebral cortex, thalamus and hypothalamus, hippocampus of diencephalons; midbrain, cerebellum). Labeling intensity was judged by two independent observers, according to the following criteria: 0, no labeling; 1, light brown labeling; 2, moderate brown labeling; 3, dark brown labeling. 2.5. Quantification of mRNA for TNF␣ and IL-1 Total RNAs were extracted from cerebral biopsies with RNAzolTM B (Duotech, Friendswood, TX, USA) and after treatment with RNase-free DNase (RQ1 DNase, promega, Madison, Wisconsin, USA) 1 g of RNA was reverse transcribed into cDNA in 20 l final volume. TNF␣ and IL-1 mRNA expression in the different group samples were normalized for -actin (primer sequence: 5 -CCAACTGGGACGATATGGAG-3 and 5 -CACAATGCCAGTGGTACGAC-3 ) cDNA content by competitive PCR (TaKaRa, Otsu, Japan) as previously described [29]. TNF␣ (primer sequence: 5 -TATGCCTCAGCCTCTTCTCC-3 and 5 -TTGAGAGACATGCCGTTGG3 ) and IL-1 (primer sequence: 5 -GACCTCCAACAAGAGCTTCCG-3 and 5 -TTGAGAGACATGCCGTTGG3 ) mRNA expression was quantified using a Quantitative Competitive PCR Kit (TaKaRa, Otsu, Japan). The results were expressed as TNF␣/-actin and IL-1/-actin ratio. 2.6. Quantification of IL-1/IL-10 by semi quantitative PCR All the samples were normalized to the same -actin concentration. Each PCR was performed coamplifing IL1/IL-10 (IL-10 primer sequence: 5 -GACCTCCAACAAGAGCTTCCG-3 and 5 -TTGAGAGACATGCCGTTGG3 ). Gels were scanned by transmission densitometry, and the areas of the peaks were calculated in arbitrary units. 2.7. Statistical analysis Survival curves were estimated by Kaplan Mayer test and their comparison was made by the long rank test; to evaluate statistically the survival curves of the positive control group and the immunized groups, a Savage Test was performed. Mann–Whitney U-test was applied to histology and immunohistochemistry; the expression of cytokines was compared by Student’s t-test.
3. Results 3.1. Immunization improves survival time after prion infection A total of 24 intraperitoneally infected animals were followed up to 216 days. General clinical features, the appearance and the development of the pathology into the groups of
G. Magri et al. / Vaccine 23 (2005) 2862–2868
2865
Table 1 Survival times Immunizing peptide
Individuals per group
Infection (i.p.) 263K, 105 ID50
Individual survival range
Survival time (days) Mean ± S.D.
None (A) PrP 105–128 (B) PrP 119–146 (C) PrP 142–179
6 6 6 6
+ + + +
111–150 109–171 112–198 110–214
139 157 165 165
± ± ± ±
24 49 43 54
Median 131 148 161 170
Survival times in hamsters (Mesocricetus auratus) immunized with synthetic oligopeptides homologous to the hamster prion protein and infected intraperitoneally with 263K scrapie agent.
animals were carefully followed up from the day of the experimental infection to point out possible differences correlated with immunization. No adverse effects were observed in the six animals treated with adjuvant alone (negative control: NC) while typical symptoms of the disease (hyperaesthesia, motor incoordination, lack of feeding capacity and lateral recumbent position) arose in the non-immunized infected controls (positive control: PC) as well as in the peptide-immunized animals. All the animals died 6–7 days after the outcome of the abovementioned symptoms and uncensored time was recorded for exclusion from the trial. Average survival time of the non-immunized infected controls (positive control: PC) was 139 ± 24 days. All the peptide-immunized animals showed a longer average survival time (group A, 157 ± 49 days; group B, 165 ± 43 days; group C, 165 ± 54 days) (Table 1). However, probably due to small groups and high variability neither the long rank test nor Savage Test showed significant statistical differences between survival curves. 3.2. Immunization induces prion-specific antibodies To test the efficacy of the immunization process we evaluated the specific seroconversion by ELISA. For this aim, we randomly sampled two animals/group at three different times (T28 , T62 , T103 ) and we analyzed peptide-specific antibodies in serum samples (Fig. 1). In all the assays performed, the
antibody levels in samples from immunized animals were far above those of background and negative controls. The response was peptide-specific as inferred by the observations that: (1) the antibody levels against free peptides were elevated in all the tested samples; (2) antibody levels in sera of the negative controls (preimmune sera, T0 ) were below the threshold of detection. Animals treated with adjuvant alone were constantly antibody negative. 3.3. Effect of immunization: histological parameters Whereas the lesion profile was comparable in all the groups examined, the intensity of spongiosis was different when vaccinated animals were compared to PC. As expected, the NC group did not show lesions in any of the areas analysed (data not shown). A decrease of lesions was seen from hindbrain to forebrain. The highest degree of spongiosis was evident in the medulla, while the hippocampus appeared unaffected. Cerebral cortex of telencephalon and cerebral cortex of diencephalon showed similar mild degrees of vacuolation. In the diencephalon, the thalamus and the hypothalamus were moderately affected. In the midbrain, both the tectum and the substantia nigra were moderately vacuolised. The cerebellar cortex of the folia was slightly affected in every group (Fig. 2). It is interesting to notice that in all the areas examined, the degree of spongiosis detected in B and C groups (the hamsters with the longest survival after infection) was lower than the one observed for PC. 3.4. Effect of immunization: immunohistochemical analysis
Fig. 1. The levels of antibody against free peptides represent the mean value between two individual animals. Peptide-specific antibodies were detected in A, B and C groups in three following samples (T28, T62, and T103). In all the assays performed, the background was always less than 0.030 OD and the negative controls were always between 0.002 OD and 0.005 OD.
Although the PrPres intensity varied slightly in some areas, the immuno labeling profile was similar in all the examined hamsters. The stronger intensity of labeling was seen in the medulla whereas the hippocampus was unaffected. The immunostaining of frontal cortex and sub-meningeal area of telencephalon was moderate, and the septal nuclei and subependymal area were mildly labeled. In the diencephalon, the cerebral cortex, the hypothalamus and the thalamus showed a similar moderate immuno-staining. The substantia nigra and the sub-ependymal area were slightly labeled. The tectum of midbrain was markedly stained in B group, while the other groups showed a lower labeling intensity (Fig. 2). It is important to underline that in each group of vaccinated animals the
2866
G. Magri et al. / Vaccine 23 (2005) 2862–2868
Fig. 2. (a) Brain lesion profile of each group of animals included in the study. The results are reported as mean values and vacuolation was scored on a scale of 0–4 in the following areas: (1) cerebral cortex of telencephalon; (2) septal nuclei; (3) cerebral cortex of diencephalons; (4) hippocampus; (5) thalamus; (6) hypothalamus; (7) tectum of midbrain; (8) substantia nigra; (9) cerebellar cortex of the folia; (10) medulla. (b) Immunostaining PrPres profile of each group of animals included in the study. The results are reported as mean values and the intensity of labeling was scored on a scale of 0–3 in the following areas: (1) cerebral cortex of telencephalon; (2) septal nuclei; (3) sub-ependymal area; (4) sub-meningeal area; (5) cerebral cortex of the diencephalons; (6) hippocampus; (7) thalamus; (8) hypothalamus; (9) tectum of midbrain; (10) substantia nigra; (11) sub-ependymal area of midbrain; (12) cerebellar corex of the folia; (13) medulla. (c) Representative scrapie lesions of four degree of severity: 1, few vacuoles; 2, several vacuoles evenly distributed; 3, moderate numbers or many vacuoles evenly distributed; 4, numerous vacuoles (haematoxylin and eosin; 10×). (d) Representative intensity of PrPres labeling: 0, no reactivity; 1, slight reactivity; 2, moderate reactivity; 3, strong reactivity. PrPres (10×).
mean score of staining intensity was lower than in PC. Again, the lowest score was seen in B and C groups. The intensity of GFAP immunoreactivity showed a trend similar to that observed for PrPres labeling. Thus, in the immunized groups, and in particular in B and C animals, the immunoreactivity was clearly lower than in PC hamsters (data not shown).
last result strongly suggests that the expression of proinflammatory and immunosuppressive cytokines is modulated by the immunization procedure.
3.5. Immunization reduces IL-1 and TNF-␣ mRNA
Prion diseases fall into the category of conformational disorders characterized by the extracellular accumulation of a constitutively expressed protein in an infectious diseaseassociated isoform [30]. Alzheimer’s disease is also characterized by the conformational change of a physiologic protein: in this case, soluble amyloid  (A) peptides accumulate as aggregated/fibrillar A [31]. Recently, immunization with A peptides has been shown to be successful at reducing cerebral amyloid accumulation in transgenic mouse models of Alzheimer’s disease. One possible mechanism of action is
The expression of proinflammatory cytokine, IL-1 and TNF␣, was reduced in all immunized groups compared to PC. These differences reached statistical significance in groups B (TNF-␣: p < 0.05) and C (TNF-␣ and IL-1, p < 0.05; Fig. 3). Furthermore, the IL-1/IL-10 ratio, quantified by semi quantitative PCR, showed a trend toward reduction in the immunized groups compared to PC. Though these differences did not reach statistical significance, this
4. Discussion
Fig. 3. Mean values and standard errors for IL-1 (a), TNF-␣ (b) and IL-1/IL-10 (c) specific mRNA obtained in each group of animals included in the study. In panels A and B, results are expressed as the ratio between IL1- or TNF-␣ and -actin mRNA; in panel C, results are expressed as the ratio between IL1- and IL-10 mRNA. PC, positive controls; NC, negative controls: A, B, C, immunized animals. p < 0.05.
G. Magri et al. / Vaccine 23 (2005) 2862–2868
that anti-A antibodies could facilitate the clearance of A by monocytic/microglial cells that are triggered via signals mediated by Fc receptors [17–19]. This observation, together with the results obtained in different prion disease models [20–23], has suggested that the induction of antibodies could play a critical role in the treatment of these degenerative diseases. We, therefore, investigated in a hamster scrapie model whether active immunization with synthetic peptides, resembling different portion of the prion protein, could influence disease onset. In none of the immunized animals, the treatment prevented the development of disease, nevertheless survival time was increased in all the hamsters that underwent immunization. The observation that elevated antibody levels were detected in the immunized animals suggests that the elicitation of humoral immunity could be critical for a successful therapeutic response. The hystopathological analyses performed on brain tissue demonstrated a decrease of the lesions in immunized animals compared to infected but hamster that were not immunized. These differences were particularly evident in B and C groups, the hamsters with the longest survival time after infection. Similarly, the immunization process was associated with a reduction of Prpres deposition mainly in the B and C groups; thus, confirming vaccination to interfere with the neuropathogenetic mechanisms involved in the brain spongiform degeneration. Another parameter considered in immunohistochemistry was GFAP, a powerful marker of astrocytosis [5]. Similarly to the previous results, GFAP level was diminished in the immunized hamsters, thus suggesting a causal correlation between PrP deposition and astrocytosis. It is not easy to explain how vaccination delays the onset of prion disease in these animals. Based on the results reported, herewith, it is possible to hypothesize a critical role of peptide-specific antibodies in the conversion of the normal cellular prion protein into the toxic one. As a matter of fact, the antibodies that are mostly effective at inhibiting PrPres formation in a dose dependent manner (monoclonal anti-PrP antibody 6H4: residues 144–152 prion protein; Fab D18: residues 132–156) recognize the same epitopes present in peptides B and C [20,21,32]. We, therefore, argue that the antibodies induced by the immunization with B and C peptides, could directly block or modify the PrP cellular interaction with PrPres . According to this hypothesis, the protective effect of antibodies was reduced in mice immunized using a peptide (peptide A) localized outside the critical site for the conversion of PrP into PrPres . Our data are partially at variance with those reported by recently published works [33,34] arguing against the efficacy of peptide immunization with or without CFA. Several differences in both the animal model, the protocol of immunization, the adjuvants and the immunogens employed might account for the differing results. On the other hand, a recent report showed that, similarly to the data presented herewith, the survival of hamsters immunized with PrP105–125 and infected by scrapie via dietary exposure is indeed prolonged [35].
2867
To better understand the molecular mechanisms involved in this disease model, and to clarify the effect of vaccination at a molecular level, we evaluated the expression of some of the mediators of inflammation (IL-1, TNF-␣, IL-10) in brain tissues. These analyses represent an innovation in the study of a vaccine approach to prion diseases, as the role of cytokines in the pathogenesis of TSE is still extremely debated [7–10,36–38]. Although prion diseases are not associated to a systemic immune response [39], some neuropathological evidences suggest that, similarly to Alzheimer’s disease, cerebral lesions could be related to a local inflammatory process orchestrated by cytokines. Results presented herewith show an increased amount of pro-inflammatory cytokines in infected (PC) compared to uninfected (NC) animals, thus supporting a role for cytokines in the onset of TSE. Additionally, the expression of IL1 and TNF␣ mRNA was significantly reduced in all the immunized compared to PC animals. To verify if vaccination would also result in an increased expression of anti-inflammatory cytokines, and because IL-10 down-regulates the generation of pro-inflammatory cytokines [40,41], we also evaluated the expression of IL-10 in all the hamsters. In the immunized animals belonging to B and C groups, an increase in IL-10 expression was observed. IL10 production at cerebral level exerts a protective action on neurons and plays an anti-apoptotic role on microglial cells [42,43]. Thus, these data contribute to clarify the protective effects of vaccination at the cerebral level. It is important to underline that pro-inflammatory cytokines were particularly reduced, IL-10 was mostly increased, and consequently, the IL1/IL10 ratio was mostly diminished, in those groups of hamsters (B and C) showing the longest survival time and the most robust reduction in the deposition of PrP and GFAP expression as well as in the degree of spongiosis. These results suggest that immunization significantly reduces inflammation. In conclusion, immunization of hamsters with peptides resembling the sequence of hamster prion protein induces a partial resistance to infection that is associated with the generation of antibodies and the reduction of PrPres deposition at cerebral level, which in turn induces a decrease in inflammation status and thus a reduction of cerebral lesions. These data, although preliminary, shed some light on the immune pathogenesis of prion diseases, and lend support to the use of immune-based approaches in the prevention of these diseases.
References [1] Prusiner SB. Molecular biology and pathogenesis of prion disease. Trends Biochem Sci 1998;21:482–7. [2] Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci 2001;24:519–50. [3] Brown DR. Microglia and prion disease. Microsc Res Tech 2001;54(2):71–80. [4] Hafiz FB, Brown DR. A model for the mechanism of astrogliosis in prion disease. Mol Cell Neurosci 2000;16(3):221–32.
2868
G. Magri et al. / Vaccine 23 (2005) 2862–2868
[5] Kordek R, Liberski PP, Yanagihara R, Isaacson S, Gajdusek CD. Molecular analysis of prion protein (PrP) and glial fibrillary acidic protein (GFAP) transcripts in experimental Creuzfeldt–Jakob disease in mice. Acta Neurobiol Exp 1997;57:85–90. [6] Prusiner SB. Prions. PNAS 1998;95:13363–83. [7] Campbell IL, Eddleston M, Kemper P, Oldstone MBA, Hobbs M. Activation of cerebral cytokine gene expression and its correlation with onset of reactive astrocyte and acute-phase response gene expression in scrapie. J Virol 1994;68:2383–7. [8] Kordek R, Nerurkar VR, Liberski PP, Isaacson S, Yanagihara R, Gajdusek DC. Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt–Jakob disease in mice. PNAS 1996;93:9754–8. [9] Williams AE, van Dam AM, Man-A-Hing WK, Berkenbosch F, Eikelenboom P, Fraser H. Cytokine, prostaglandins and lipocortin1 are present in the brains of scrapie-infected mice. Brain Res 1994;654:200–6. [10] Kim JI, Ju WK, Choi JH, et al. Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapieinfected mice. Brain Res Mol Brain Res 1999;73:17–27. [11] Eikelenboom P, Bate C, Van Gool WA, et al. Neuroinflammation in Alzheimer’s disease and prion disease. Glia 2002;40:232–9. [12] Forloni G, Tagliavini F, Bugiani O, Salmona M. Amyloid in Alzheimer’s disease and prion-related encephalopathies: studies with synthetic peptides. Prog Neurobiol 1996;49:287–315. [13] Checler F, Vincent B. Alzheimer’s and prion diseases: distinct pathologies, common proteolytic denominators. Trends Neurosci 2002;25(12):616–20. [14] Wisniewski T, Brown DR, Sigurdsson EM. Therapeutics in Alzheimer’s and prion diseases. Biochem Soc Trans 2002;30(4):574–8. [15] Aguzzi A, Glatzel M, Montrasio F, Prinz M, Heppner FL. Interventional strategies against prion diseases. Nat Rev Neurosci 2001;2(10):745–9. [16] Heppner FL, Arrighi I, Kalinke U, Aguzzi A. Immunity against prions? Trends Mol Med 2001;7(11):477–9. [17] Schenk D, Barbour R, Dunn W, et al. Immunization with amyloidbeta attenuates Alzheimer disease-like pathology in the PDAPP mouse. Nature 1999;400:173–7. [18] Janus C, Pearson J, McLaurin J, et al. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease. Nature 2000;408(6815):979–82. [19] Bard F, Cannon C, Barbour R, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 2000;6(8):916–9. [20] Enari M, Flechsig E, Weissmann C. Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. PNAS (USA) 2001;98:9295–9. [21] Peretz D, Williamson RA, Kaneko K, et al. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 2001;412:739–42. [22] Heppner FL, Musahl C, Arrighi I, et al. Prevention of scrapie pathogenesis by transgenic expression of anti prion protein antibodies. Science 2001;294:178–82. [23] Souan L, Tal Y, Felling Y, Cohen IR, Taraboulos A, Mor F. Modulation of proteinase-K resistant prion protein by prion peptide immunization. Eur J Immunol 2001;31:2338–46.
[24] Fields GB, Noble RL. Solid phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids. Int J Peptide Protein Res 1990;35:161–214. [25] King DS, Fields CG, Fields GB. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Peptide Protein Res 1990;42:255–66. [26] Liu FT, Zinnecker M, Hamaoka T, Katz DH. Procedures for preparation and isolation of protein and Synthetic copolymer of d-amino acids and immunochemical characterization of such conjugates. Biochemistry 1979;18:690–7. [27] Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001;2(10):734–44. [28] Hetz C, Soto C. Protein misfolding and disease: the case of prion disorders. Cell Mol Life Sci 2003;60(1):133–43. [29] Xi YG, Cardone F, Pocchiari M. Detection of proteinase resistant protein (PrP) in small brain tissue samples from Creutzfeldt–Jakob disease patients. J Neurol Sci 1994;124:171–3. [30] Trabattoni D, Saresella M, Biasin M, et al. B7-H1 is up-regulated in HIV infection and is a novel surrogate marker of disease progression. Blood 2002;101(7):2514–20. [31] Holtzman DM. A beta conformational change is central to Alzheimer’s disease. Neurobiol Aging 2002;23(6):1085–8. [32] Arbel M, Lavie V, Solomon BJ. Generation of antibodies against prion protein in wild-type mice via helix 1 peptide immunization. Neuroimmunology 2003;144(1–2):38–45. [33] Tal Y, Souan L, Cohen IR, et al. Complete Freund’s adjuvant immunization prolongs survival in experimental prion disease in mice. J Neurosci Res 2003;71(2):286–90. [34] Rosset MB, Ballerini C, Gregoire S, et al. Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice. J Immunol 2004;172(9):5168–74. [35] Schwarz A, Kratke O, Burwinkel M, et al. Immunization with a synthetic prion derived peptide prolongs survival time of mice orally exposed to the scrapie agent. Neurosci Lett 2003;350:187–9. [36] Peyrin JM, Lasmezas CI, Haik S, et al. Microglial cells respond to amyloidogenic PrP peptide by the production of inflammatory cytokines. Neuroreport 1999;10:723–9. [37] Perry VH, Cunningham C, Boche D. A typical inflammation in the central nervous system in prion disease. Curr Opin Neurol 2002;15(3):349–54. [38] Cunningham C, Boche D, Perry VH. Transforming growth factor beta1, the dominant cytokine in murine prion disease: influence on inflammatory cytokine synthesis and alteration of vascular extracellular matrix. Neuropathol Appl Neurobiol 2002;28(2):107– 19. [39] Aucouturier P, Frangione B, Wisniewski T. Prion diseases and the immune system. Ann Med Intern 1999;150(2):75–8 (Paris). [40] Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy—review of a new approach. Pharmacol Rev 2003;55(2):241–9. [41] Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–91. [42] Strle K, Zhou JH, Shen WH. Interleukin-10 in the brain. Crit Rev Immunol 2001;21(5):427–49. [43] Fraser H, Dickinson AG. The sequential development of the brain lesions of scrapie in three strains of mice. J Comp Pathol 1968;78:301–11.
Decrease in pathology and progression of scrapie after immunisation with synthetic prion protein peptides in hamsters夽 Giuliana Magria , Mario Clericia,∗ , Paola Dall’Arab , Mara Biasina , Maria Caramellic , Cristina Casalonec , Maria Laura Gianninob , Renato Longhid , Luca Piacentinia , Silvia Della Bellae , Paola Gazzuolac , Piera Anna Martinob , Silvia Della Bellaa , Claudia Pollerab , Maria Puricellib , Francesco Servidab , Ines Crescioc , Adriano Boassoa , Wilma Pontib , Giorgio Polib a
Laboratory of Immunology, DSP LITA Vialba, Universita’ degli Studi di Milano, Via G.B. Grassi 74, 20157 Milano, Italy b Dipartimento di Patologia Animale, Igiene e Sanit` a Pubblica Veterinaria, Sezione di Microbiologia e Immunologia, Universit`a degli Studi di Milano, Centro di Eccellenza sulle Malattie Neurodegenerative, Milan, Italy c Centro per lo Studio e le Ricerche sulle Encefalopatie Animali e Neuropatologie Comparate, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle D’Aosta, Torino, Italy d Istituto di Biocatalisi e Riconoscimento Molecolare, C.N.R., Milano, Italy e The Immunology Department of Biomedical Sciences and Technologies, University of Milan, Milan, Italy Received 10 May 2004; received in revised form 12 October 2004; accepted 25 November 2004 Available online 31 December 2004
Abstract Effective therapy for prion diseases is currently unavailable. Recently, vaccination was shown to be effective in mouse models of a particular neurodegenerative conditions: Alzheimer’s disease (AD). Here, we report that vaccination with synthetic oligopeptides homologous to the hamster (Mesocricetus auratus) prion protein augments survival time in animals infected intraperitoneally with 263K scrapie agent. For each hamster included in the study, prion-specific serum antibodies as well as deposition of pathological prion protein (PrPres ), glial fibrillary acidic protein (GFAP), and mRNA expression for cytokines (TNF␣, IL-1, IL-10) in brain tissues were evaluated. In immunized animals, increased survival after challenge was associated with a reduction of cerebral lesion, PrP deposition and GFAP expression; in these animals, anti-prion protein peptide antibody levels were increased, and the expression of pro-inflammatory cytokines (TNF␣ and IL-1) was reduced. Vaccination could be an effective therapeutic approach to postpone disease onset. © 2004 Elsevier Ltd. All rights reserved. Keywords: Prion; Vaccination; Inflammation
1. Introduction Prion diseases, or transmissible spongiform encephalopathies (TSE), are rare, progressive, fatal neurode夽 Supported by grants from ISS: “Programma Nazionale di Ricerca sull’
AIDS” and “Progetti strategici: fattori genetici, patogenetici e biochimici responsabili della sensibilit`a/resistenza alle EST”, by Centro di Eccellenza CISI, and by the Japan Health Science Foundation. ∗ Corresponding author. Tel.: +39 02 5031 9679; fax: +39 02 5031 9677. E-mail address: [email protected] (M. Clerici). 0264-410X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2004.11.067
generative conditions that include Creutzfeldt–Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats [1,2]. These diseases are characterized by spongiform degeneration of the brain, accompanied by appearance of activated microglia and astrocytes as shown by the altered expression of the glial fibrillary acidic protein (GFAP), a marker of astrocytosis [3–5]. The hallmark of TSE diseases is the conversion of normal cellular prion protein (PrPc ) into an infectious disease-associated protease-resistant isoform (PrPres ) that accumulates primarily in the brain
G. Magri et al. / Vaccine 23 (2005) 2862–2868
and in the lymphoreticular system [6]. PrP deposits are also associated with a chronic inflammatory response mainly based on cytokine production (TNF-␣, IL-1, IL-6) [7–10]. Prion diseases share important clinical, neuropathological, and cell biological features with another, more common degenerative cerebral disease. Both AD and prion diseases are characterized by a cascade of events that terminate with the extracellular deposition of amyloid- (A) or PrPres fibrillar peptides, respectively [11–14]. Currently, none of the prion diseases has an effective treatment [15]. In spite of this, recent evidences suggest that immunotherapeutical intervention could be worth pursuing [16]. It has recently been shown that immunization of transgenic mice with AD-related neuropathology using fibrillar A1–42 as an antigen, reduces or prevents cerebral A amyloid deposits [17,18]. An antibody-mediated response is likely to be critical for a therapeutic response, since analog results have been obtained with passive immunization [19]. Similar approaches for prion diseases have recently shown that: (1) anti-PrP monoclonal antibodies or fragment antigen binding (Fab) with little or no affinity for PrPres can inhibit PrPres formation in a dose-dependent manner [20,21]; (2) coexpression of anti-PrPc antibodies (6H4) and PrPc in transgenic mice (Prnp+/− -6H4) does not induce an obvious autoimmune disease and confers protection from scrapie [22]; and (3) immunization of wild-type mice with prion protein peptides induces anti-PrP antibodies and reduces PrPres formation in scrapie-infected neuroblastomas transplanted into immunized mice [23]. We investigated whether active immunization with synthetic peptides of prion protein could influence the progression of prion disease in a hamster model of scrapie.
2. Material and methods 2.1. Conjugate preparation, vaccination and infection protocol Thirty Female Golden Syrian hamsters, 3–4 weeks old (Charles River, Como, Italy) were used according to the EU International Guidelines for animal experimentation. The animals were randomly divided into three groups (six animals/group). A negative control group of animals (NC) treated with adjuvant alone and a positive control group (PC) (infected but non-immunized animals) were also included in the study. Three peptides (peptide A: p105–128: cPKTNMKHMAGAAAAGAVVGGLGGY; peptide B: p119–146: cGAVVGGLGGYMLGSAMSRPMMHFGNDWE; peptide C: p142– 179: GNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNFVHDC) homologous to the hamster prion protein and resembling different fragments (30–32 amino acids) of the molecule were used. The choice of these three peptides was made because they cover the N-terminal (A peptide)
2863
and the central (B and C peptides) sequence of hamster PrP. In particular, B and C peptides contains the protein domain possibly involved in PrP conversion process. Peptides were synthesized by the solid phase Fmoc method [24] using an Applied Biosystem model 433A Peptide Synthesizer (Foster City, CA, USA). To the peptides not incorporating thiolfunction, cysteine was added at the amino termini or at the carboxy termini of the peptide to enable specific conjugation to the carrier protein. After peptide assembly, peptides were cleaved from the resin by treatment with a mixture of 80% trifluoroacetic acid, 5% water, 5% phenol, 5% thioanisole, 2.5% ethandithiol and 2.5% triisopropylsilane (reagent K) [25] for 3 h at room temperature, with concomitant side chain deprotection. Peptides were lyophilized and than analyzed and purified to apparent homogeneity by reversed phase high performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis of the peptides was performed on a Voyager-RP Biospectrometry Workstation (PE Biosystem Inc.). Observed experimental values for peptide masses were in agreement with theoretical calculated values. Peptides were specifically conjugated to freshly prepared maleimido-activated Mariculture Keyhole Limpet Hemocyanin (mcKLH), using sulfosuccimnimidyl 4(N-maleimidomethyl)cyclohexane-1-carboxylate (SulfoSMCC), both from Pierce, according with the procedure described by Liu et al. [26], diluted in pyrogen free saline solution mixed with an equal volume of complete (first vaccination) or incomplete (booster vaccinations) Freund’s adjuvant immediately before administration. Animals were inoculated distributing 0.5 ml of the suspension containing a total amount of 10 g of different peptides both by intramuscular route into right and left popliteus and by subcutaneous and intradermic route in the right and left dorsal region. Animals were immunized with three injections at 10 days interval before infection (T0, T11, T20), challenged by intraperitoneal route with 105 ID50 of 263K hamster adapted scrapie strain (T25) and then immunized with other five injections at 20 days interval post-infection (T28, T62, T81, T103, T126). 2.2. Antibody response ELISA 96-well microplates (Maxisorp, Nunc, Roskilde, Denmark) were coated for 18 h at 4 ◦ C with 100 l/well of free peptides, diluted in carbonate–bicarbonate buffer at the concentration of 250 ng/ml. After four washings with PBS-Tween 0.05% (PBS-T), 100 l/well of 1:100 pre-diluted serum samples were added and the plates incubated for 1 h at 37 ◦ C. After four PBS-T washings, 100 l/well of 1:500 prediluted horseradish peroxidase-conjugated anti-hamster IgG (ICN Biomedicals, Cappel, Irvine, CA, USA) were added and the plates incubated for 1 h at 37 ◦ C. After four more
2864
G. Magri et al. / Vaccine 23 (2005) 2862–2868
PBS-T washings, 100 l of ABTS substrate was added to each well. The reaction was stopped by the addition of 50 l of 1% SDS/well and the plates were read at 405 nm using the Labsystem Multiskan EX (Helsinki, Finland). 2.3. Histological examination The whole brain was collected. Each specimen was cut in half longitudinally along the midline; one half was fixed in 10% formol saline and decontaminated with 96% formic acid for 1 h. The unfixed portion was frozen at −80 ◦ C. Four transverse sections were obtained from each specimen, paraffin embedded, cut in serial sections (5–6 m), and stained with haematoxylin and eosin by standard procedures. Ten areas of the grey matter were examined in four neuroanatomical locations (cerebral cortex septal nuclei of telencephalon; cerebral cortex, hypothalamus, thalamus, hippocampus of diencephalon; tectum of midbrain and substantia nigra of mesencephalon; cerebellar cortex of the folia and medulla) [27]. The lesion profile method was applied to each area, by two independent observers, giving a score from 0 to 4 depending on the density of vacuolation and spongiosis. 2.4. Immunohistochemical analysis: PrPres and GFAP staining Five-micrometer thick tissue sections were de-waxed and re-hydrated by routine methods. For PrPres , the sections were immersed in 98% formic acid for 20 min, washed in distilled water, and autoclaved for 20 min at 121 ◦ C in distilled water. Endogenous peroxidase activity was blocked in 3% hydrogen peroxide for 20 min at room temperature. For gfap endogenous peroxidase activity was blocked in 3% hydrogen peroxide for 20 min at room temperature and subjected to an antigen retrieval procedure with two 5 min 850 W microwave oven passages in 0.01 M (pH 6) citrate buffer. To remove non-specific tissue antigens, the sections were incubated with 5% normal goat serum for 20 min at room temperature. Incubation with the primary antibody 1/IST [28] raised in rabbit against 263K Scrapie strain, diluted 1:250, and anti-gfap policlonal antibody (Dako, Carpinteria, CA) diluted 1:1000 was performed overnight at 4 ◦ C. The rest of the immunohistochemical procedure was carried out by a commercial immunoperoxidase technique (Vectastain ABC kit, Vector Burlingame, CA), using 3,3 diaminobenzidine (Dako, Carpinteria, CA) as chromogen and the sections were counterstained with Meyer’s haematoxylin. For PrPres , 13 areas in four neuroanatomical location were examined (cerebral cortex, sub-ependymal area, submeningeal area and septal nuclei of telencephalon; cerebral cortex, hypothalamus, thalamus, hippocampus of diencephalon; tectum of midbrain, sub-ependymal area, and substantia nigra of mesencephalon; cerebellar cortex of the folia and medulla); for GFAP, seven sites in four neuroanatomical
area were examined (cerebral cortex of telencephalon; cerebral cortex, thalamus and hypothalamus, hippocampus of diencephalons; midbrain, cerebellum). Labeling intensity was judged by two independent observers, according to the following criteria: 0, no labeling; 1, light brown labeling; 2, moderate brown labeling; 3, dark brown labeling. 2.5. Quantification of mRNA for TNF␣ and IL-1 Total RNAs were extracted from cerebral biopsies with RNAzolTM B (Duotech, Friendswood, TX, USA) and after treatment with RNase-free DNase (RQ1 DNase, promega, Madison, Wisconsin, USA) 1 g of RNA was reverse transcribed into cDNA in 20 l final volume. TNF␣ and IL-1 mRNA expression in the different group samples were normalized for -actin (primer sequence: 5 -CCAACTGGGACGATATGGAG-3 and 5 -CACAATGCCAGTGGTACGAC-3 ) cDNA content by competitive PCR (TaKaRa, Otsu, Japan) as previously described [29]. TNF␣ (primer sequence: 5 -TATGCCTCAGCCTCTTCTCC-3 and 5 -TTGAGAGACATGCCGTTGG3 ) and IL-1 (primer sequence: 5 -GACCTCCAACAAGAGCTTCCG-3 and 5 -TTGAGAGACATGCCGTTGG3 ) mRNA expression was quantified using a Quantitative Competitive PCR Kit (TaKaRa, Otsu, Japan). The results were expressed as TNF␣/-actin and IL-1/-actin ratio. 2.6. Quantification of IL-1/IL-10 by semi quantitative PCR All the samples were normalized to the same -actin concentration. Each PCR was performed coamplifing IL1/IL-10 (IL-10 primer sequence: 5 -GACCTCCAACAAGAGCTTCCG-3 and 5 -TTGAGAGACATGCCGTTGG3 ). Gels were scanned by transmission densitometry, and the areas of the peaks were calculated in arbitrary units. 2.7. Statistical analysis Survival curves were estimated by Kaplan Mayer test and their comparison was made by the long rank test; to evaluate statistically the survival curves of the positive control group and the immunized groups, a Savage Test was performed. Mann–Whitney U-test was applied to histology and immunohistochemistry; the expression of cytokines was compared by Student’s t-test.
3. Results 3.1. Immunization improves survival time after prion infection A total of 24 intraperitoneally infected animals were followed up to 216 days. General clinical features, the appearance and the development of the pathology into the groups of
G. Magri et al. / Vaccine 23 (2005) 2862–2868
2865
Table 1 Survival times Immunizing peptide
Individuals per group
Infection (i.p.) 263K, 105 ID50
Individual survival range
Survival time (days) Mean ± S.D.
None (A) PrP 105–128 (B) PrP 119–146 (C) PrP 142–179
6 6 6 6
+ + + +
111–150 109–171 112–198 110–214
139 157 165 165
± ± ± ±
24 49 43 54
Median 131 148 161 170
Survival times in hamsters (Mesocricetus auratus) immunized with synthetic oligopeptides homologous to the hamster prion protein and infected intraperitoneally with 263K scrapie agent.
animals were carefully followed up from the day of the experimental infection to point out possible differences correlated with immunization. No adverse effects were observed in the six animals treated with adjuvant alone (negative control: NC) while typical symptoms of the disease (hyperaesthesia, motor incoordination, lack of feeding capacity and lateral recumbent position) arose in the non-immunized infected controls (positive control: PC) as well as in the peptide-immunized animals. All the animals died 6–7 days after the outcome of the abovementioned symptoms and uncensored time was recorded for exclusion from the trial. Average survival time of the non-immunized infected controls (positive control: PC) was 139 ± 24 days. All the peptide-immunized animals showed a longer average survival time (group A, 157 ± 49 days; group B, 165 ± 43 days; group C, 165 ± 54 days) (Table 1). However, probably due to small groups and high variability neither the long rank test nor Savage Test showed significant statistical differences between survival curves. 3.2. Immunization induces prion-specific antibodies To test the efficacy of the immunization process we evaluated the specific seroconversion by ELISA. For this aim, we randomly sampled two animals/group at three different times (T28 , T62 , T103 ) and we analyzed peptide-specific antibodies in serum samples (Fig. 1). In all the assays performed, the
antibody levels in samples from immunized animals were far above those of background and negative controls. The response was peptide-specific as inferred by the observations that: (1) the antibody levels against free peptides were elevated in all the tested samples; (2) antibody levels in sera of the negative controls (preimmune sera, T0 ) were below the threshold of detection. Animals treated with adjuvant alone were constantly antibody negative. 3.3. Effect of immunization: histological parameters Whereas the lesion profile was comparable in all the groups examined, the intensity of spongiosis was different when vaccinated animals were compared to PC. As expected, the NC group did not show lesions in any of the areas analysed (data not shown). A decrease of lesions was seen from hindbrain to forebrain. The highest degree of spongiosis was evident in the medulla, while the hippocampus appeared unaffected. Cerebral cortex of telencephalon and cerebral cortex of diencephalon showed similar mild degrees of vacuolation. In the diencephalon, the thalamus and the hypothalamus were moderately affected. In the midbrain, both the tectum and the substantia nigra were moderately vacuolised. The cerebellar cortex of the folia was slightly affected in every group (Fig. 2). It is interesting to notice that in all the areas examined, the degree of spongiosis detected in B and C groups (the hamsters with the longest survival after infection) was lower than the one observed for PC. 3.4. Effect of immunization: immunohistochemical analysis
Fig. 1. The levels of antibody against free peptides represent the mean value between two individual animals. Peptide-specific antibodies were detected in A, B and C groups in three following samples (T28, T62, and T103). In all the assays performed, the background was always less than 0.030 OD and the negative controls were always between 0.002 OD and 0.005 OD.
Although the PrPres intensity varied slightly in some areas, the immuno labeling profile was similar in all the examined hamsters. The stronger intensity of labeling was seen in the medulla whereas the hippocampus was unaffected. The immunostaining of frontal cortex and sub-meningeal area of telencephalon was moderate, and the septal nuclei and subependymal area were mildly labeled. In the diencephalon, the cerebral cortex, the hypothalamus and the thalamus showed a similar moderate immuno-staining. The substantia nigra and the sub-ependymal area were slightly labeled. The tectum of midbrain was markedly stained in B group, while the other groups showed a lower labeling intensity (Fig. 2). It is important to underline that in each group of vaccinated animals the
2866
G. Magri et al. / Vaccine 23 (2005) 2862–2868
Fig. 2. (a) Brain lesion profile of each group of animals included in the study. The results are reported as mean values and vacuolation was scored on a scale of 0–4 in the following areas: (1) cerebral cortex of telencephalon; (2) septal nuclei; (3) cerebral cortex of diencephalons; (4) hippocampus; (5) thalamus; (6) hypothalamus; (7) tectum of midbrain; (8) substantia nigra; (9) cerebellar cortex of the folia; (10) medulla. (b) Immunostaining PrPres profile of each group of animals included in the study. The results are reported as mean values and the intensity of labeling was scored on a scale of 0–3 in the following areas: (1) cerebral cortex of telencephalon; (2) septal nuclei; (3) sub-ependymal area; (4) sub-meningeal area; (5) cerebral cortex of the diencephalons; (6) hippocampus; (7) thalamus; (8) hypothalamus; (9) tectum of midbrain; (10) substantia nigra; (11) sub-ependymal area of midbrain; (12) cerebellar corex of the folia; (13) medulla. (c) Representative scrapie lesions of four degree of severity: 1, few vacuoles; 2, several vacuoles evenly distributed; 3, moderate numbers or many vacuoles evenly distributed; 4, numerous vacuoles (haematoxylin and eosin; 10×). (d) Representative intensity of PrPres labeling: 0, no reactivity; 1, slight reactivity; 2, moderate reactivity; 3, strong reactivity. PrPres (10×).
mean score of staining intensity was lower than in PC. Again, the lowest score was seen in B and C groups. The intensity of GFAP immunoreactivity showed a trend similar to that observed for PrPres labeling. Thus, in the immunized groups, and in particular in B and C animals, the immunoreactivity was clearly lower than in PC hamsters (data not shown).
last result strongly suggests that the expression of proinflammatory and immunosuppressive cytokines is modulated by the immunization procedure.
3.5. Immunization reduces IL-1 and TNF-␣ mRNA
Prion diseases fall into the category of conformational disorders characterized by the extracellular accumulation of a constitutively expressed protein in an infectious diseaseassociated isoform [30]. Alzheimer’s disease is also characterized by the conformational change of a physiologic protein: in this case, soluble amyloid  (A) peptides accumulate as aggregated/fibrillar A [31]. Recently, immunization with A peptides has been shown to be successful at reducing cerebral amyloid accumulation in transgenic mouse models of Alzheimer’s disease. One possible mechanism of action is
The expression of proinflammatory cytokine, IL-1 and TNF␣, was reduced in all immunized groups compared to PC. These differences reached statistical significance in groups B (TNF-␣: p < 0.05) and C (TNF-␣ and IL-1, p < 0.05; Fig. 3). Furthermore, the IL-1/IL-10 ratio, quantified by semi quantitative PCR, showed a trend toward reduction in the immunized groups compared to PC. Though these differences did not reach statistical significance, this
4. Discussion
Fig. 3. Mean values and standard errors for IL-1 (a), TNF-␣ (b) and IL-1/IL-10 (c) specific mRNA obtained in each group of animals included in the study. In panels A and B, results are expressed as the ratio between IL1- or TNF-␣ and -actin mRNA; in panel C, results are expressed as the ratio between IL1- and IL-10 mRNA. PC, positive controls; NC, negative controls: A, B, C, immunized animals. p < 0.05.
G. Magri et al. / Vaccine 23 (2005) 2862–2868
that anti-A antibodies could facilitate the clearance of A by monocytic/microglial cells that are triggered via signals mediated by Fc receptors [17–19]. This observation, together with the results obtained in different prion disease models [20–23], has suggested that the induction of antibodies could play a critical role in the treatment of these degenerative diseases. We, therefore, investigated in a hamster scrapie model whether active immunization with synthetic peptides, resembling different portion of the prion protein, could influence disease onset. In none of the immunized animals, the treatment prevented the development of disease, nevertheless survival time was increased in all the hamsters that underwent immunization. The observation that elevated antibody levels were detected in the immunized animals suggests that the elicitation of humoral immunity could be critical for a successful therapeutic response. The hystopathological analyses performed on brain tissue demonstrated a decrease of the lesions in immunized animals compared to infected but hamster that were not immunized. These differences were particularly evident in B and C groups, the hamsters with the longest survival time after infection. Similarly, the immunization process was associated with a reduction of Prpres deposition mainly in the B and C groups; thus, confirming vaccination to interfere with the neuropathogenetic mechanisms involved in the brain spongiform degeneration. Another parameter considered in immunohistochemistry was GFAP, a powerful marker of astrocytosis [5]. Similarly to the previous results, GFAP level was diminished in the immunized hamsters, thus suggesting a causal correlation between PrP deposition and astrocytosis. It is not easy to explain how vaccination delays the onset of prion disease in these animals. Based on the results reported, herewith, it is possible to hypothesize a critical role of peptide-specific antibodies in the conversion of the normal cellular prion protein into the toxic one. As a matter of fact, the antibodies that are mostly effective at inhibiting PrPres formation in a dose dependent manner (monoclonal anti-PrP antibody 6H4: residues 144–152 prion protein; Fab D18: residues 132–156) recognize the same epitopes present in peptides B and C [20,21,32]. We, therefore, argue that the antibodies induced by the immunization with B and C peptides, could directly block or modify the PrP cellular interaction with PrPres . According to this hypothesis, the protective effect of antibodies was reduced in mice immunized using a peptide (peptide A) localized outside the critical site for the conversion of PrP into PrPres . Our data are partially at variance with those reported by recently published works [33,34] arguing against the efficacy of peptide immunization with or without CFA. Several differences in both the animal model, the protocol of immunization, the adjuvants and the immunogens employed might account for the differing results. On the other hand, a recent report showed that, similarly to the data presented herewith, the survival of hamsters immunized with PrP105–125 and infected by scrapie via dietary exposure is indeed prolonged [35].
2867
To better understand the molecular mechanisms involved in this disease model, and to clarify the effect of vaccination at a molecular level, we evaluated the expression of some of the mediators of inflammation (IL-1, TNF-␣, IL-10) in brain tissues. These analyses represent an innovation in the study of a vaccine approach to prion diseases, as the role of cytokines in the pathogenesis of TSE is still extremely debated [7–10,36–38]. Although prion diseases are not associated to a systemic immune response [39], some neuropathological evidences suggest that, similarly to Alzheimer’s disease, cerebral lesions could be related to a local inflammatory process orchestrated by cytokines. Results presented herewith show an increased amount of pro-inflammatory cytokines in infected (PC) compared to uninfected (NC) animals, thus supporting a role for cytokines in the onset of TSE. Additionally, the expression of IL1 and TNF␣ mRNA was significantly reduced in all the immunized compared to PC animals. To verify if vaccination would also result in an increased expression of anti-inflammatory cytokines, and because IL-10 down-regulates the generation of pro-inflammatory cytokines [40,41], we also evaluated the expression of IL-10 in all the hamsters. In the immunized animals belonging to B and C groups, an increase in IL-10 expression was observed. IL10 production at cerebral level exerts a protective action on neurons and plays an anti-apoptotic role on microglial cells [42,43]. Thus, these data contribute to clarify the protective effects of vaccination at the cerebral level. It is important to underline that pro-inflammatory cytokines were particularly reduced, IL-10 was mostly increased, and consequently, the IL1/IL10 ratio was mostly diminished, in those groups of hamsters (B and C) showing the longest survival time and the most robust reduction in the deposition of PrP and GFAP expression as well as in the degree of spongiosis. These results suggest that immunization significantly reduces inflammation. In conclusion, immunization of hamsters with peptides resembling the sequence of hamster prion protein induces a partial resistance to infection that is associated with the generation of antibodies and the reduction of PrPres deposition at cerebral level, which in turn induces a decrease in inflammation status and thus a reduction of cerebral lesions. These data, although preliminary, shed some light on the immune pathogenesis of prion diseases, and lend support to the use of immune-based approaches in the prevention of these diseases.
References [1] Prusiner SB. Molecular biology and pathogenesis of prion disease. Trends Biochem Sci 1998;21:482–7. [2] Collinge J. Prion diseases of humans and animals: their causes and molecular basis. Annu Rev Neurosci 2001;24:519–50. [3] Brown DR. Microglia and prion disease. Microsc Res Tech 2001;54(2):71–80. [4] Hafiz FB, Brown DR. A model for the mechanism of astrogliosis in prion disease. Mol Cell Neurosci 2000;16(3):221–32.
2868
G. Magri et al. / Vaccine 23 (2005) 2862–2868
[5] Kordek R, Liberski PP, Yanagihara R, Isaacson S, Gajdusek CD. Molecular analysis of prion protein (PrP) and glial fibrillary acidic protein (GFAP) transcripts in experimental Creuzfeldt–Jakob disease in mice. Acta Neurobiol Exp 1997;57:85–90. [6] Prusiner SB. Prions. PNAS 1998;95:13363–83. [7] Campbell IL, Eddleston M, Kemper P, Oldstone MBA, Hobbs M. Activation of cerebral cytokine gene expression and its correlation with onset of reactive astrocyte and acute-phase response gene expression in scrapie. J Virol 1994;68:2383–7. [8] Kordek R, Nerurkar VR, Liberski PP, Isaacson S, Yanagihara R, Gajdusek DC. Heightened expression of tumor necrosis factor alpha, interleukin 1 alpha, and glial fibrillary acidic protein in experimental Creutzfeldt–Jakob disease in mice. PNAS 1996;93:9754–8. [9] Williams AE, van Dam AM, Man-A-Hing WK, Berkenbosch F, Eikelenboom P, Fraser H. Cytokine, prostaglandins and lipocortin1 are present in the brains of scrapie-infected mice. Brain Res 1994;654:200–6. [10] Kim JI, Ju WK, Choi JH, et al. Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapieinfected mice. Brain Res Mol Brain Res 1999;73:17–27. [11] Eikelenboom P, Bate C, Van Gool WA, et al. Neuroinflammation in Alzheimer’s disease and prion disease. Glia 2002;40:232–9. [12] Forloni G, Tagliavini F, Bugiani O, Salmona M. Amyloid in Alzheimer’s disease and prion-related encephalopathies: studies with synthetic peptides. Prog Neurobiol 1996;49:287–315. [13] Checler F, Vincent B. Alzheimer’s and prion diseases: distinct pathologies, common proteolytic denominators. Trends Neurosci 2002;25(12):616–20. [14] Wisniewski T, Brown DR, Sigurdsson EM. Therapeutics in Alzheimer’s and prion diseases. Biochem Soc Trans 2002;30(4):574–8. [15] Aguzzi A, Glatzel M, Montrasio F, Prinz M, Heppner FL. Interventional strategies against prion diseases. Nat Rev Neurosci 2001;2(10):745–9. [16] Heppner FL, Arrighi I, Kalinke U, Aguzzi A. Immunity against prions? Trends Mol Med 2001;7(11):477–9. [17] Schenk D, Barbour R, Dunn W, et al. Immunization with amyloidbeta attenuates Alzheimer disease-like pathology in the PDAPP mouse. Nature 1999;400:173–7. [18] Janus C, Pearson J, McLaurin J, et al. A beta peptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease. Nature 2000;408(6815):979–82. [19] Bard F, Cannon C, Barbour R, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med 2000;6(8):916–9. [20] Enari M, Flechsig E, Weissmann C. Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody. PNAS (USA) 2001;98:9295–9. [21] Peretz D, Williamson RA, Kaneko K, et al. Antibodies inhibit prion propagation and clear cell cultures of prion infectivity. Nature 2001;412:739–42. [22] Heppner FL, Musahl C, Arrighi I, et al. Prevention of scrapie pathogenesis by transgenic expression of anti prion protein antibodies. Science 2001;294:178–82. [23] Souan L, Tal Y, Felling Y, Cohen IR, Taraboulos A, Mor F. Modulation of proteinase-K resistant prion protein by prion peptide immunization. Eur J Immunol 2001;31:2338–46.
[24] Fields GB, Noble RL. Solid phase peptide synthesis utilizing 9fluorenylmethoxycarbonyl amino acids. Int J Peptide Protein Res 1990;35:161–214. [25] King DS, Fields CG, Fields GB. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int J Peptide Protein Res 1990;42:255–66. [26] Liu FT, Zinnecker M, Hamaoka T, Katz DH. Procedures for preparation and isolation of protein and Synthetic copolymer of d-amino acids and immunochemical characterization of such conjugates. Biochemistry 1979;18:690–7. [27] Allan SM, Rothwell NJ. Cytokines and acute neurodegeneration. Nat Rev Neurosci 2001;2(10):734–44. [28] Hetz C, Soto C. Protein misfolding and disease: the case of prion disorders. Cell Mol Life Sci 2003;60(1):133–43. [29] Xi YG, Cardone F, Pocchiari M. Detection of proteinase resistant protein (PrP) in small brain tissue samples from Creutzfeldt–Jakob disease patients. J Neurol Sci 1994;124:171–3. [30] Trabattoni D, Saresella M, Biasin M, et al. B7-H1 is up-regulated in HIV infection and is a novel surrogate marker of disease progression. Blood 2002;101(7):2514–20. [31] Holtzman DM. A beta conformational change is central to Alzheimer’s disease. Neurobiol Aging 2002;23(6):1085–8. [32] Arbel M, Lavie V, Solomon BJ. Generation of antibodies against prion protein in wild-type mice via helix 1 peptide immunization. Neuroimmunology 2003;144(1–2):38–45. [33] Tal Y, Souan L, Cohen IR, et al. Complete Freund’s adjuvant immunization prolongs survival in experimental prion disease in mice. J Neurosci Res 2003;71(2):286–90. [34] Rosset MB, Ballerini C, Gregoire S, et al. Breaking immune tolerance to the prion protein using prion protein peptides plus oligodeoxynucleotide-CpG in mice. J Immunol 2004;172(9):5168–74. [35] Schwarz A, Kratke O, Burwinkel M, et al. Immunization with a synthetic prion derived peptide prolongs survival time of mice orally exposed to the scrapie agent. Neurosci Lett 2003;350:187–9. [36] Peyrin JM, Lasmezas CI, Haik S, et al. Microglial cells respond to amyloidogenic PrP peptide by the production of inflammatory cytokines. Neuroreport 1999;10:723–9. [37] Perry VH, Cunningham C, Boche D. A typical inflammation in the central nervous system in prion disease. Curr Opin Neurol 2002;15(3):349–54. [38] Cunningham C, Boche D, Perry VH. Transforming growth factor beta1, the dominant cytokine in murine prion disease: influence on inflammatory cytokine synthesis and alteration of vascular extracellular matrix. Neuropathol Appl Neurobiol 2002;28(2):107– 19. [39] Aucouturier P, Frangione B, Wisniewski T. Prion diseases and the immune system. Ann Med Intern 1999;150(2):75–8 (Paris). [40] Asadullah K, Sterry W, Volk HD. Interleukin-10 therapy—review of a new approach. Pharmacol Rev 2003;55(2):241–9. [41] Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A. Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–91. [42] Strle K, Zhou JH, Shen WH. Interleukin-10 in the brain. Crit Rev Immunol 2001;21(5):427–49. [43] Fraser H, Dickinson AG. The sequential development of the brain lesions of scrapie in three strains of mice. J Comp Pathol 1968;78:301–11.